Peptide screen

ABSTRACT

The invention relates to methods to screen for agents that interact with member of the vitronectrin receptor family, αvβ3 integrin and agents obtainable by said methods.

[0001] The invention relates to methods to screen for agents that interact with a member of the vitronectin receptor family, αvβ3 integrin.

[0002] Angiogenesis, the development of new blood vessels from an existing vascular bed, is a complex multistep process that involves the degradation of components of the extracellular matrix and then the migration, proliferation and differentiation of endothelial cells to form tubules and eventually new vessels. Angiogenesis is important in normal physiological processes including, by example and not by way of limitation, embryo implantation; embryogenesis and development; and wound healing. Excessive angiogenesis is also involved in pathological conditions such as tumour cell growth and non-cancerous conditions such as neovascular glaucoma, rheumatoid arthritis, psoriasis and diabetic retinopathy.

[0003] The vascular endothelium is normally quiescent. However upon activation endothelial cells proliferate and migrate to form microtubules which will ultimately form a capillary bed to supply blood to developing tissues and, of course, a growing tumour. A number of growth factors has been identified which promote/activate endothelial cells to undergo angiogenesis. These include, by example and not by way of limitation; vascular endothelial growth factor (VEGF); transforming growth factor (TGFb); acidic and basic fibroblast growth factor (aFGF and bFGF); and platelet derived growth factor (PDGF) (1,2).

[0004] VEGF is a an endothelial cell-specific growth factor which has a very specific site of action, namely the promotion of endothelial cell proliferation, migration and differentiation. VEGF is a dimeric complex comprising two identical 23 kDa polypeptides. The monomeric form of VEGF can exist as four distinct polypeptides of different molecular weights, each being derived from an alternatively spliced mRNA. Of the four monomeric forms, two exist as membrane bound VEGF and two are soluble. VEGF is expressed by a wide variety of cell/tissue types including embryonal tissues; proliferating keratinocytes; macrophages; tumour cells. Studies (2) have shown VEGF is highly expressed in many tumour cell-lines including glioma and AIDS-associated Karposi's sarcoma VEGF activity is mediated through VEGF specific receptors expressed by endothelial cells and tumour cells. Indeed, VEGF receptors are up-regulated in endothelial cells which infiltrate tumours thereby promoting tumour cell growth.

[0005] bFGF is a growth factor which functions to stimulate the proliferation of fibroblasts and endothelial cells. bFGF is a single polypeptide chain with a molecular weight of 16.5 kDa. Several molecular forms of bFGF have been discovered which differ in the length of their amino terminal region. However the biological functions of the various molecular forms appears to be the same. bFGF is produced by the pituitary gland and is encoded by a single gene located on human chromosome 4.

[0006] A number of endogenous inhibitors of angiogenesis have been discovered, examples of which are angiostatin and endostatin, which are formed by the proteolytic cleavage of plasminogen and collagen XVIII respectively. Both of these factors have been shown to suppress the activity of pro-angiogenic growth factors such as vascular VEGF and bFGF. Both also suppress endothelial cell responses to VEGF and bFGF in vitro, and reduce the vascularisation and growth of experimental tumours in animal models.

[0007] A potent, new inhibitor of angiogenesis, which is a 50 kDa proteolytic fragment of fibrinogen, fibrinogen E, is disclosed in our co-pending application WO01/88129, which is incorporated by reference. Furthermore, we have identified a domain within the fibrinogen E fragment which has the same anti-angiogenic activity as the very much larger fibrinogen E fragment. The domain is disclosed in co-pending patent application WO02/18440, which is incorporated by reference. The domain is located at the amino terminus of the α chain and is referred to as α1-24.

[0008] We now disclose the receptor to which these polypeptides bind to mediate the anti-angiogenic effect. The peptides bind to and inhibit the activity of the vitronectin receptor, αvβ3 integrin. The vitronectin receptor is a member of a large family of receptors generally referred to as integrins.

[0009] It is known that the vitronectin receptor is involved in mediating many cellular processes. Basement membranes are organised as thin layers of specialised extracellular matrix that provide support for epithelial and endothelial cells. Basement membranes provide both mechanical support and regulate cellular behaviour such as differentiation, proliferation and migration of cells such as endothelial cells.

[0010] The αvβ3 integrin binds to numerous extracellular matrix proteins; for example, fibrinogen, fibronectin, osteopontin, thrombospondin, vitronectin and von Willebrand factor, largely via interaction with a tripeptide sequence found in the matrix proteins, arginine-glycine-aspartic acid (‘RGD’) A few other extracellular matrix proteins such as tumstatin, a collagen fragment derived from the NC1 domain of the α3 chain of collagen IV, bind to αvβ3 via a non-RGD-dependent mechanism. Maeshima et al 2000 and Maeshima et al 2001 describe a region of tumstatin which binds αvβ3 which is found between amino acids 54-132. This region does not have an RGD motif Furthermore, tumstatin binding blocks angiogenesis mediated by activated endothelial cells.

[0011] In our co-pending application (WO02/18440) we described an anti-angiogenic peptide referred to as α1-24 which lacks a canonical RGD motif and therefore binds αvβ3 in an RGD-independent fashion. The identification of this peptide which binds αvβ3 will allow the identification of agents which inhibit the binding of α1-24 and therefore identify agents with the potential to block extracellular matrix: αvβ3 interactions via this novel, non-RGD interaction pathway. The identified agents will have utility with respect to blocking angiogenesis thereby ameliorating disease conditions which depend on angiogenesis, for example wound healing, cancer.

[0012] It is an object of the invention to provide a method for the identification of agents which interfere with the interaction of a fibrinogen E fragment, or peptide derivatives thereof, and the vitronectin receptor.

[0013] It is a further object of the invention to provide a method for the identification of agents which bind the vitronectin receptor wherein the agents have angiogenesis modulatory activity.

[0014] According to an aspect of the invention there is provided a screening method for the identification of agents which modulate the interaction of a fibrinogen E fragment, or peptide derivatives thereof, with the vitronectin receptor.

[0015] In a preferred method of the invention said method comprises the steps of:

[0016] i) providing a polypeptide comprising the amino acid sequence presented in FIG. 2, or active binding fragment thereof;

[0017] ii) providing at least one peptide comprising an amino acid sequence selected from the sequences presented in FIG. 1;

[0018] iii) providing at least one agent to be tested;

[0019] iv) forming a preparation of (i), (ii) and (iii); and

[0020] v) detecting or measuring the effect of the agent in (iii) on the interaction of the polypeptide and peptide in (i) and (ii).

[0021] In a further preferred method of the invention said agent is pre-incubated with polypeptide in (i) prior to addition of the peptide in (ii).

[0022] In an alternative preferred method of the invention said agent is pre-incubated with the peptide in (ii) prior to addition to the polypeptide in (i).

[0023] In a preferred method of the invention the peptide in (ii) comprises an amino acid sequence, or part thereof, consisting of the sequence: XXXXXLXEXXGXXXPRVXXR

[0024] In a yet further preferred method of the invention the peptide in (ii) comprises an amino acid sequence, or part thereof, consisting of the sequence: SXXXXXLXEXXGXXXPRVXXR

[0025] In a yet further preferred method of the invention said peptide comprises an amino acid sequence as represented by the sequence: GEGXFLXEXXGXXXPRVVXR

[0026] In a yet further preferred method of the invention said peptide comprises an amino acid sequence as represented by the sequence: GEG XFL XXX XXXXX XXXX XX.

[0027] X is any amino acid residue selected from the group consisting of alanine, valine, leucine, isoleucine, or proline. Preferably X is alanine.

[0028] In a preferred embodiment of the invention said peptide comprises an amino acid sequence as represented by the sequences presented in table 1. Preferably said peptides comprising said sequence have anti-angiogenic activity.

[0029] In yet a further preferred embodiment of the invention the peptide comprises an amino acid sequence as represented by the overlapping part of two fragments presented in table 1. In a more preferred embodiment, said peptide is derived from the overlapping part of the peptides AHI-401 and AHI-378 in table 1. Preferably, said peptide derived from the overlapping part of the peptides AHI-401 and AHI-378 comprises one additional amino acid residue at the N-terminus.

[0030] Thus, in a preferred method of the invention said peptide comprises an amino acid sequence as represented by the sequence: XFLAEGGGVXG

[0031] X is any amino acid residue selected from the group consisting of A, R, N, D, C, E, Q, G, H, I, L, K, M, F, P, S, T, W, Y, V. Preferably X is selected from the group consisting of A, V, L, I and P, more preferably X is a basic amino acid selected from the group consisting of H, R and K, or an acidic amino acid selected from the group consisting of D and E.

[0032] In another preferred embodiment, the N-terminal X is selected from the group consisting of D and E, whereas the C-terminal X is selected from the group consisting of H, R and K, or alternatively, the N-terminal amino acid is selected from the group consisting of H, R and K, whereas the C-terminal X is selected from the group consisting of D and E. In a particularly preferred embodiment, the N-terminal X is D and the C-terminal X is R.

[0033] In a yet further preferred method of the invention said peptide comprises an amino acid sequence selected from the group consisting of: GEG DFL AEG GGV RGP RVVE R GEG DFL AEG GGX RGP RVVE R GEG DFL AEG GGV XGP RVVE R GEG DFL AEG GGV RXP RVVE R GEG DFL AEG GGV RGP RVXE R GEG DFL AEG GGV RGP RVVXR GEG DFL AEG GGXXXP RVVXR GEG DFL AEG GGXXXP RVXXR.

[0034] X is any amino acid residue selected from the group consisting of alanine, valine, leucine, isoleucine, or proline. Preferably X is alanine.

[0035] In a further preferred method of the invention said part thereof is represented by the amino acid sequence from +1 to +15 of the amino acid sequence: SXXXXXLXEXXGXXXPRVXXR

[0036] In a further preferred method of the invention said part thereof is represented by the amino acid sequence from +6 to +21 of the amino acid sequence: XXXXXLXEXXGXXXPRVXXR

[0037] In a yet further preferred method of the invention said part thereof is represented by the amino acid sequence +6 to +15 of the amino acid sequence XXXXXLXEXXGXXXPRVXXR

[0038] In a further preferred method of the invention said peptide consists of the peptide amino acid sequences as herein disclosed.

[0039] In a further preferred embodiment of the invention said peptide is 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or 24 amino acid residues in length and comprises an amino acid sequence according to the invention.

[0040] In a preferred method of the invention the peptide in (ii) is selected from the group consisting of: 1 A X S XXX DFLA X GGGV XX P X V V XX H 2 ADSGEGDFLAXGGGVRGPRV VE X H 3 ADSGEGDFLA X GGGVRGPRV VERH 4 ADSGEGDFLA EGGGV X GPRV VERH 5 ADSGEG X FLA EGGGVRGPRV VERH 6 A X SG X GDFLA EGGGVRGPRV VERH 7 ADSG X GDFLA EGGGVRGPRV VERH 8 A X SGEGDFLA EGGGVROPRV VERH 9 ADSGEGDFLA EGGGVRGPRV V X RH

[0041] wherein X is any amino acid residue.

[0042] Preferably X is a hydrophobic amino acid residue and more preferably is selected from the group consisting of alanine, valine, leucine, isoleucine, or proline. Most preferably said amino acid residue is alanine.

[0043] In a further preferred method of the invention the vitronectin receptor is presented in a soluble form or by a cell. Preferably said cell naturally expresses the vitronectin receptor. Examples of cells which naturally express the vitronectin receptor are endothelial cells, smooth muscle cells, osteoclasts and tumour cells. Alternatively said cell does not naturally express the vitronectin receptor, in which case said cells are preferably genetically engineered to express the vitronectin receptor.

[0044] According to a further aspect of the invention there are provided agent(s) identified by the screening method according to the invention. Preferably said agent(s) interfere with the interaction of peptides or polypeptides with vitronectin receptor binding activity. Alternatively said agent promotes the interaction of polypeptides with vitronectin receptor binding activity.

[0045] In a preferred embodiment of the invention said agent is a foldamer, i.e. a polymer with a strong tendency to adopt a specific, compact conformation (15), that modulates the binding of α1-24 with the αvβ3 integrin.

[0046] In a more preferred embodiment of the invention said agent is a peptide, peptide derivative or low molecular weight agent that modulates the binding of α1-24 with the αvβ3 integrin.

[0047] In a further preferred embodiment of the invention said agent is a polypeptide. Preferably said agent is an antibody.

[0048] In a further preferred embodiment of the invention said antibody is a monoclonal antibody. Preferably said monoclonal antibody is a chimeric antibody. Alternatively said antibody is a humanised antibody.

[0049] Antibodies, also known as immunoglobulins, are protein molecules which have specificity for foreign molecules (antigens). Immunoglobulins (Ig) are a class of structurally related proteins consisting of two pairs of polypeptide chains, one pair of light (L) (low molecular weight) chain (κ or λ), and one pair of heavy (H) chains (γ, α, μ, δ and ε), all four linked together by disulphide bonds. Both H and L chains have regions that contribute to the binding of antigen and that are highly variable from one Ig molecule to another. In addition, H and L chains contain regions that are non-variable or constant.

[0050] The L chains consist of two domains. The carboxy-terminal domain is essentially identical among L chains of a given type and is referred to as the “constant” (C) region. The amino terminal domain varies from L chain to L chain and contributes to the binding site of the antibody. Because of its variability, it is referred to as the “variable” (V) region.

[0051] The H chains of Ig molecules are of several classes, α, μ, σ, α, and γ (of which there are several sub-classes). An assembled Ig molecule consisting of one or more units of two identical H and L chains, derives its name from the H chain that it possesses. Thus, there are five Ig isotypes: IgA, IgM, IgD, IgE and IgG (with four sub-classes based on the differences in the H chains, i.e., IgG1, IgG2, IgG3 and IgG4). Further detail regarding antibody structure and their various functions can be found in, Using Antibodies: A laboratory manual, Cold Spring Harbour Laboratory Press.

[0052] Chimeric antibodies are recombinant antibodies in which all of the V-regions of a mouse or rat antibody are combined with human antibody C-regions. Humanised antibodies are recombinant hybrid antibodies which fuse the complementarity-determining regions from a rodent antibody V-region with the framework regions from the human antibody V-regions. The C-regions from the human antibody are also used. The complementarity-determining regions (CDRs) are the regions within the N-terminal domain of both the heavy and light chain of the antibody to where the majority of the variation of the V-region is restricted. These regions form loops at the surface of the antibody molecule. These loops provide the binding surface between the antibody and antigen.

[0053] Antibodies from non-human animals provoke an immune response to the foreign antibody and its removal from the circulation. Both chimeric and humanised antibodies have reduced antigenicity when injected to a human subject because there is a reduced amount of rodent (i.e. foreign) antibody within the recombinant hybrid antibody, while the human antibody regions do not illicit an immune response. This results in a weaker immune response and a decrease in the clearance of the antibody.

[0054] According to another aspect of the invention there is provided a screening method for the identification of agents with vitronectin binding activity comprising the steps of:

[0055] i) providing a member of the integrin family which binds the peptide α1-24, or variant thereof;

[0056] ii) providing at least one candidate binding agent;

[0057] iii) forming a preparation comprising a combination of (i) and (ii);

[0058] iv) detecting or measuring the binding of the agent in (ii) with the polypeptide in (i); and optionally

[0059] v) testing the capability of the agent to modulate angiogenesis.

[0060] In a preferred method of the invention an agent with vitronectin receptor-binding activity has anti-angiogenic activity. Alternatively, the agent with vitronectin receptor binding activity has pro-angiogenic activity.

[0061] In a preferred method of the invention the integrin is the vitronectin receptor and is αvβ3 integrin. Preferably, the integrin is represented by the amino acid sequence as shown in FIG. 2, or fragment thereof.

[0062] In a further preferred method of the invention said vitronectin receptor is in a soluble form or is presented by a cell.

[0063] In a further preferred method of the invention said cell naturally expresses a vitronectin receptor.

[0064] The vitronectin receptor αvβ3 integrin is expressed by a variety of cell-types, for example, endothelial cells, smooth muscle cells, osteoclasts and tumour cells.

[0065] Preferably said cell is an endothelial cell.

[0066] In an alternative embodiment of the invention said cell does not naturally express a vitronectin receptor. Preferably said cell is genetically engineered to express the vitronectin receptor.

[0067] According to a further aspect of the invention there is provided an agent obtainable by the method according to the invention. Preferably the agent has anti-angiogenic activity. Alternatively, said agent has pro-angiogenic activity.

[0068] In a preferred embodiment of the invention the agent is a foldamer, i.e. a polymer with a strong tendency to adopt a specific, compact conformation (15). In a more preferred embodiment of the invention the agent is a low molecular weight compound, a peptide, peptide derivative or variant, or a polypeptide.

[0069] An embodiment of the invention will now be described, by example only, and with reference to the following figures:

[0070]FIG. 1 represents the amino acid sequence of the α1-24 polypeptide and α1-24 variants of fibrinogen E;

[0071]FIG. 2 is the amino acid sequence of a member of vitronectin receptor family, αvβ3 integrin (α chain and β chain;

[0072]FIG. 3 represents the blocking effect of anti-vitronectin receptor polyclonal antibodies on α1-24 anti-angiogenic activity; and

[0073]FIG. 4 represents a binding assay using immobilised. αvβ3 integrin incubated with α1-24 peptide or vitronectin.

[0074] Table 1 represents a summary of the anti-angiogenic activity of polypeptides as herein disclosed.

[0075] Materials and Methods

[0076] Human integrin αvβ3 was obtained from Chemicon International Inc (28835 Single Oak Drive Temecula, CA92590, USA, catalogue code CC1018.

[0077] Direct Binding Assay for α_(v)β₃ Integrin

[0078] 1. Coat plates overnight with test protein (α1-24, modified α1-24, or vitronectin) in PBS at room temperature in sealed plates.

[0079] 2. Wash three times in washing buffer (0.1% BSA, 0.05% Tween-20 in PBS)

[0080] 3. Block with 1% BSA (in PBS) for 30 minutes at 37° C.

[0081] 4. Wash three times with washing buffer

[0082] 5. Add an equimolar amount of α_(v)β₃ integrin (Chemicon) for 1 hour at 37° C.

[0083] 6. Wash three times with washing buffer

[0084] 7. Add monoclonal antibody to α_(v)β₃ integrin (Chemicon) 10 μg/ml for 1 hour at 37° C.

[0085] 8. Wash three times with washing buffer

[0086] 9. Add secondary antibody conjugated to HRP (Dako) diluted 1:1000 in PBS for an hour at 37° C.

[0087] 10. Wash three times with washing buffer

[0088] 11. Add substrate—Tetramethylbenzidine (100 μg/ml) (Sigma)

[0089] 12. After colour development (15-30 minutes) read on an ELISA plate reader at 630 nm.

[0090] Cell Culture.

[0091] Adult human dermal microvascular endothelial cells (HuDMECs) were obtained commercially (TCS Biologicals, Buckinghamshire, United Kingdom) and cultured in microvascular endothelial cell growth medium (EGM). This medium contains heparin (10 ng/ml), hydrocortisone, human epidermal growth factor (10 ng/ml), human fibroblast growth factor (10 ng/ml) (such endothelial growth factors are necessary for routine passaging of HuDMECs in culture) and dibutyryl cyclic AMP. This was supplemented with 5% heat-inactivated FCS, 50 μg/ml gentamicin and 50 ng/ml amphotericin B (TCS Biologicals, United Kingdom). Murine endothelial cells (SVEC 4-10) were obtained from the ATCC and cultured in DMEM+10% FCS. Cells were grown at 37° C. in a 100% humidified incubator with a gas phase of 5% CO₂ and routinely screened for mycoplasma. Prior to their use in the assays indicated below, HuDMECs were grown to 80% confluency, incubated in DMEM+1% FCS for 2 h, then harvested with 0.05% trypsin solution, washed twice and resuspended to the cell density required for each assay (see below).

[0092] α1-24 Modified Peptides.

[0093] The α1-24 peptides were generated by standard peptide synthesis methods using Fmoc amino acids and a synthesis machine. The purity of the peptide was checked by mass spectroscopy.

[0094] Tubule Formation Assay.

[0095] 24 well plates were coated with 30 μl/well of growth factor-reduced (GF-reduced) Matrigel (Becton Dickinson Labware, Bedford, Mass.). Endothelial cells plated on this matrix migrate and differentiate into tubules within 6 h of plating as described previously (14). HuDMECs or SVEC 4-10 cells were seeded at a density of 4×10⁴ cells/ml and incubated for 6 h in 500 μl of either DMEM+1% FCS alone (control), or this medium ±10 ng/ml VEGF or bFGF in the presence or absence of α1-24 peptides. Assessment of tubule formation involved fixing the cell preparation in 70% ethanol at 4° C. for 15 minutes, rinsing in PBS and staining with haematoxylin and eosin. Three random fields of view in 3 replicate wells for each test condition were visualised under low power (×40 magnification), and colour images captured using a Fuji digital camera linked to a Pentium III computer (containing a frame grabber board). Tubule formation was assessed by counting the number of tubule branches and the total area covered by tubules in each field of view using image analysis software supplied by Scion Image.

[0096] Migration Assay

[0097] The Boyden chamber technique was adapted from (13) and used to evaluate HuDMEC migration across a porous membrane towards a concentration gradient of either VEGF (10 ng/ml) or bFGF (10 ng/ml). The Neuro Probe 48 well microchemotaxis chamber (Neuro Probe Inc, Cabin John, Md.) was used with 8 μm pore size polycarbonate membranes (Neuro Probe Inc, Cabin John, Md.) coated with 100 μg/ml collagen type IV. 10 ng/ml VEGF or bFGF alone or with various concentrations of α1-24 peptides were dissolved in DMEM+1% FCS and placed in the lower wells. The collagen-coated membrane was then placed over this and 50 μl of 25×10⁴ HuDMECs/ml (in DMEM containing 1% FCS) added to the upper chamber. The chambers were then incubated at 37° C. for 4.5 h. The chamber was then dismantled, the membrane removed and non-migrated cells scraped off the upper surface. Migrated cells on the lower surface were fixed with methanol, stained with Hema ‘Gurr’ rapid staining kit (Merck, Leics, United Kingdom) and counted using a light microscope (×160 magnification) in 3 random fields per well. Each test condition was carried out in 3-6 replicate wells and each experiment repeated 3 times.

[0098] Proliferation Assay

[0099] The MTT (3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) assay was used as previously described (12) to assess HuDMEC proliferation induced by VEGF or bFGF in the absence or presence of α1-24 peptides. HuDMEC were seeded at 3×10³ cells/100 μl in DMEM+1% FCS±10 ng/ml VEGF or bFGF in test solution into 96 well microtitre plate for 4.5 and 6 h. At these time points, a quarter volume of MTT solution (2 mg MTT/ml PBS) was added to each well and each plate was incubated for 4 h at 37° C. resulting in an insoluble purple formazan product. The medium was aspirated and the precipitates dissolved in 100 μl DMSO buffered at pH 10.5. The absorbance was then read at 540 nm on a Dynex ELISA plate reader.

[0100] Cytotoxicity Assay

[0101] HuDMECs were seeded at a density of 1-2×10⁵ cells per well in a 24 well-plate in the absence or presence of α1-24 peptides. After 6 h, both live (following removal by trysinisation) and dead (floating) cells were harvested and cell viability of all cells present assessed using propidium iodide staining of 5000 cells in each of triplicate samples per treatment using a FACScan (Becton Dickinson) equipped with a blue laser excitation of 15 mW at 488 nm. The data was collected and analysed using Cell Quest software (Becton Dickinson).

[0102] Tumour Cell Culture

[0103] The CT26 cell fine was maintained by in vitro passage in Dulbecco's Minimal Eagles Medium containing 10% foetal calf serum, and 1% penicillin and streptomycin and maintained at 37° C. in humidified atmosphere of 5% CO₂ in air. The cell line was routinely checked to ensure freedom from mycoplasma (Mycoplasma rapid detection system, Gena-Probe Incorporated, U.S.A.)

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= any amino acid 7 Ala Xaa Ser Gly Xaa Gly Asp Phe Leu Ala Glu Gly Gly Gly Val Arg 1 5 10 15 Gly Pro Arg Val Val Glu Arg His 20 8 24 PRT Homo sapiens MISC_FEATURE X = any amino acid 8 Ala Asp Ser Gly Xaa Gly Asp Phe Leu Ala Glu Gly Gly Gly Val Arg 1 5 10 15 Gly Pro Arg Val Val Glu Arg His 20 9 24 PRT Homo sapiens MISC_FEATURE X = any amino acid 9 Ala Xaa Ser Gly Glu Gly Asp Phe Leu Ala Glu Gly Gly Gly Val Arg 1 5 10 15 Gly Pro Arg Val Val Glu Arg His 20 10 24 PRT Homo sapiens MISC_FEATURE X = any amino acid 10 Ala Asp Ser Gly Glu Gly Asp Phe Leu Ala Glu Gly Gly Gly Val Arg 1 5 10 15 Gly Pro Arg Val Val Xaa Arg His 20 11 20 PRT Homo sapiens MISC_FEATURE X = any amino acid 11 Gly Glu Gly Asp Phe Leu Ala Glu Gly Gly Gly Val Arg Gly Pro Arg 1 5 10 15 Val Val Glu Arg 20 12 20 PRT Homo sapiens MISC_FEATURE X = any amino acid 12 Gly Glu Gly Asp Phe Leu Ala Glu Gly Gly Gly Xaa Arg Gly Pro Arg 1 5 10 15 Val Val Glu Arg 20 13 20 PRT Homo sapiens MISC_FEATURE X = any amino acid 13 Gly Glu Gly Asp Phe Leu Ala Glu Gly Gly Gly Val Xaa Gly Pro Arg 1 5 10 15 Val Val Glu Arg 20 14 20 PRT Homo sapiens MISC_FEATURE X = any amino acid 14 Gly Glu Gly Asp Phe Leu Ala Glu Gly Gly Gly Val Arg Xaa Pro Arg 1 5 10 15 Val Val Glu Arg 20 15 20 PRT Homo sapiens MISC_FEATURE X = any amino acid 15 Gly Glu Gly Asp Phe Leu Ala Glu Gly Gly Gly Val Arg Gly Pro Arg 1 5 10 15 Val Xaa Glu Arg 20 16 20 PRT Homo sapiens MISC_FEATURE X = any amino acid 16 Gly Glu Gly Asp Phe Leu Ala Glu Gly Gly Gly Val Arg Gly Pro Arg 1 5 10 15 Val Val Xaa Arg 20 17 20 PRT Homo sapiens MISC_FEATURE X = any amino acid 17 Gly Glu Gly Asp Phe Leu Ala Glu Gly Gly Gly Xaa Xaa Xaa Pro Arg 1 5 10 15 Val Val Xaa Arg 20 18 20 PRT Homo sapiens MISC_FEATURE X = any amino acid 18 Gly Glu Gly Asp Phe Leu Ala Glu Gly Gly Gly Xaa Xaa Xaa Pro Arg 1 5 10 15 Val Xaa Xaa Arg 20 19 21 PRT Homo sapiens MISC_FEATURE X = any amino acid 19 Ser Xaa Xaa Xaa Xaa Xaa Leu Xaa Glu Xaa Xaa Gly Xaa Xaa Xaa Pro 1 5 10 15 Arg Val Xaa Xaa Arg 20 20 20 PRT Homo sapiens MISC_FEATURE X = any amino acid 20 Xaa Xaa Xaa Xaa Xaa Leu Xaa Glu Xaa Xaa Gly Xaa Xaa Xaa Pro Arg 1 5 10 15 Val Xaa Xaa Arg 20 21 20 PRT Homo sapiens MISC_FEATURE X = any amino acid 21 Xaa Xaa Xaa Xaa Xaa Leu Xaa Glu Xaa Xaa Gly Xaa Xaa Xaa Pro Arg 1 5 10 15 Val Val Xaa Arg 20 22 20 PRT Homo sapiens MISC_FEATURE X = any amino acid 22 Gly Glu Gly Xaa Phe Leu Xaa Glu Xaa Xaa Gly Xaa Xaa Xaa Pro Arg 1 5 10 15 Val Val Xaa Arg 20 23 20 PRT Homo sapiens MISC_FEATURE X = any amino acid 23 Gly Glu Gly Xaa Phe Leu Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1 5 10 15 Xaa Xaa Xaa Xaa 20 24 11 PRT Homo sapiens MISC_FEATURE X = any amino acid 24 Xaa Phe Leu Ala Glu Gly Gly Gly Xaa Arg Gly 1 5 10 25 24 PRT Homo sapiens 25 Ala Asp Ser Gly Glu Gly Asp Phe Leu Ala Glu Gly Gly Gly Val Arg 1 5 10 15 Gly Pro Arg Val Val Glu Arg His 20 26 24 PRT Homo sapiens 26 Ala Ala Ser Gly Glu Gly Asp Phe Leu Ala Glu Gly Gly Gly Val Arg 1 5 10 15 Gly Pro Arg Val Val Glu Arg His 20 27 24 PRT Homo sapiens 27 Ala Asp Ala Gly Glu Gly Asp Phe Leu Ala Glu Gly Gly Gly Val Arg 1 5 10 15 Gly Pro Arg Val Val Glu Arg His 20 28 24 PRT Homo sapiens 28 Ala Asp Ser Ala Glu Gly Asp Phe Leu Ala Glu Gly Gly Gly Val Arg 1 5 10 15 Gly Pro Arg Val Val Glu Arg His 20 29 24 PRT Homo sapiens 29 Ala Asp Ser Gly Ala Gly Asp Phe Leu Ala Glu Gly Gly Gly Val Arg 1 5 10 15 Gly Pro Arg Val Val Glu Arg His 20 30 24 PRT Homo sapiens 30 Ala Asp Ser Gly Glu Ala Asp Phe Leu Ala Glu Gly Gly Gly Val Arg 1 5 10 15 Gly Pro Arg Val Val Glu Arg His 20 31 24 PRT Homo sapiens 31 Ala Asp Ser Gly Glu Gly Ala Phe Leu Ala Glu Gly Gly Gly Val Arg 1 5 10 15 Gly Pro Arg Val Val Glu Arg His 20 32 24 PRT Homo sapiens 32 Ala Asp Ser Gly Glu Gly Asp Ala Leu Ala Glu Gly Gly Gly Val Arg 1 5 10 15 Gly Pro Arg Val Val Glu Arg His 20 33 24 PRT Homo sapiens 33 Ala Asp Ser Gly Glu Gly Asp Phe Ala Ala Glu Gly Gly Gly Val Arg 1 5 10 15 Gly Pro Arg Val Val Glu Arg His 20 34 24 PRT Homo sapiens 34 Ala Asp Ser Gly Glu Gly Asp Phe Leu Ala Ala Gly Gly Gly Val Arg 1 5 10 15 Gly Pro Arg Val Val Glu Arg His 20 35 24 PRT Homo sapiens 35 Ala Asp Ser Gly Glu Gly Asp Phe Leu Ala Glu Ala Gly Gly Val Arg 1 5 10 15 Gly Pro Arg Val Val Glu Arg His 20 36 24 PRT Homo sapiens 36 Ala Asp Ser Gly Glu Gly Asp Phe Leu Ala Glu Gly Ala Gly Val Arg 1 5 10 15 Gly Pro Arg Val Val Glu Arg His 20 37 24 PRT Homo sapiens 37 Ala Asp Ser Gly Glu Gly Asp Phe Leu Ala Glu Gly Gly Ala Val Arg 1 5 10 15 Gly Pro Arg Val Val Glu Arg His 20 38 24 PRT Homo sapiens 38 Ala Asp Ser Gly Glu Gly Asp Phe Leu Ala Glu Gly Gly Gly Ala Arg 1 5 10 15 Gly Pro Arg Val Val Glu Arg His 20 39 24 PRT Homo sapiens 39 Ala Asp Ser Gly Glu Gly Asp Phe Leu Ala Glu Gly Gly Gly Val Ala 1 5 10 15 Gly Pro Arg Val Val Glu Arg His 20 40 24 PRT Homo sapiens 40 Ala Asp Ser Gly Glu Gly Asp Phe Leu Ala Glu Gly Gly Gly Val Arg 1 5 10 15 Ala Pro Arg Val Val Glu Arg His 20 41 24 PRT Homo sapiens 41 Ala Asp Ser Gly Glu Gly Asp Phe Leu Ala Glu Gly Gly Gly Val Arg 1 5 10 15 Gly Ala Arg Val Val Glu Arg His 20 42 24 PRT Homo sapiens 42 Ala Asp Ser Gly Glu Gly Asp Phe Leu Ala Glu Gly Gly Gly Val Arg 1 5 10 15 Gly Pro Ala Val Val Glu Arg His 20 43 24 PRT Homo sapiens 43 Ala Asp Ser Gly Glu Gly Asp Phe Leu Ala Glu Gly Gly Gly Val Arg 1 5 10 15 Gly Pro Arg Ala Val Glu Arg His 20 44 24 PRT Homo sapiens 44 Ala Asp Ser Gly Glu Gly Asp Phe Leu Ala Glu Gly Gly Gly Val Arg 1 5 10 15 Gly Pro Arg Val Ala Glu Arg His 20 45 24 PRT Homo sapiens 45 Ala Asp Ser Gly Glu Gly Asp Phe Leu Ala Glu Gly Gly Gly Val Arg 1 5 10 15 Gly Pro Arg Val Val Glu Arg His 20 46 24 PRT Homo sapiens 46 Ala Asp Ser Gly Glu Gly Asp Phe Leu Ala Glu Gly Gly Gly Val Arg 1 5 10 15 Gly Pro Arg Val Val Glu Arg His 20 47 24 PRT Homo sapiens 47 Ala Asp Ser Gly Glu Gly Asp Phe Leu Ala Glu Gly Gly Gly Val Arg 1 5 10 15 Gly Pro Arg Val Val Glu Arg His 20 48 24 PRT Homo sapiens 48 Ala Asp Ser Gly Glu Gly Asp Phe Leu Ala Glu Gly Gly Gly Val Arg 1 5 10 15 Gly Pro Arg Val Val Glu Arg His 20 49 24 PRT Homo sapiens 49 Ala Asp Ser Gly Glu Gly Asp Phe Leu Ala Glu Gly Gly Gly Val Arg 1 5 10 15 Gly Pro Arg Val Val Glu Arg His 20 50 15 PRT Homo sapiens 50 Ala Asp Ser Gly Glu Gly Asp Phe Leu Ala Glu Gly Gly Gly Val 1 5 10 15 51 15 PRT Homo sapiens 51 Asp Ser Gly Glu Gly Asp Phe Leu Ala Glu Gly Gly Gly Val Arg 1 5 10 15 52 15 PRT Homo sapiens 52 Ser Gly Glu Gly Asp Phe Leu Ala Glu Gly Gly Gly Val Arg Gly 1 5 10 15 53 15 PRT Homo sapiens 53 Gly Glu Gly Asp Phe Leu Ala Glu Gly Gly Gly Val Arg Gly Pro 1 5 10 15 54 15 PRT Homo sapiens 54 Glu Gly Asp Phe Leu Ala Glu Gly Gly Gly Val Arg Gly Pro Arg 1 5 10 15 55 15 PRT Homo sapiens 55 Gly Asp Phe Leu Ala Glu Gly Gly Gly Val Arg Gly Pro Arg Val 1 5 10 15 56 15 PRT Homo sapiens 56 Asp Phe Leu Ala Glu Gly Gly Gly Val Arg Gly Pro Arg Val Val 1 5 10 15 57 15 PRT Homo sapiens 57 Phe Leu Ala Glu Gly Gly Gly Val Arg Gly Pro Arg Val Val Glu 1 5 10 15 58 15 PRT Homo sapiens 58 Leu Ala Glu Gly Gly Gly Val Arg Gly Pro Arg Val Val Glu Arg 1 5 10 15 59 15 PRT Homo sapiens 59 Ala Glu Gly Gly Gly Val Arg Gly Pro Arg Val Val Glu Arg His 1 5 10 15 60 16 PRT Homo sapiens 60 Ala Asp Ser Gly Glu Gly Asp Phe Leu Ala Glu Gly Gly Gly Val Arg 1 5 10 15 61 28 PRT Homo sapiens 61 Ala Asp Ser Gly Glu Gly Asp Phe Leu Ala Glu Gly Gly Gly Val Arg 1 5 10 15 Gly Pro Arg Val Val Glu Arg His Gln Ser Ala Cys 20 25 62 28 PRT Homo sapiens 62 Ala Asp Ser Gly Glu Gly Asp Phe Leu Ala Glu Gly Gly Gly Val Arg 1 5 10 15 Gly Pro Arg Val Val Glu Arg His Gln Ser Ala Cys 20 25 63 23 PRT Homo sapiens 63 Asp Ser Gly Glu Gly Asp Phe Leu Ala Glu Gly Gly Gly Val Arg Gly 1 5 10 15 Pro Arg Val Val Glu Arg His 20 64 22 PRT Homo sapiens 64 Ser Gly Glu Gly Asp Phe Leu Ala Glu Gly Gly Gly Val Arg Gly Pro 1 5 10 15 Arg Val Val Glu Arg His 20 65 21 PRT Homo sapiens 65 Gly Glu Gly Asp Phe Leu Ala Glu Gly Gly Gly Val Arg Gly Pro Arg 1 5 10 15 Val Val Glu Arg His 20 66 20 PRT Homo sapiens 66 Glu Gly Asp Phe Leu Ala Glu Gly Gly Gly Val Arg Gly Pro Arg Val 1 5 10 15 Val Glu Arg His 20 67 19 PRT Homo sapiens 67 Gly Asp Phe Leu Ala Glu Gly Gly Gly Val Arg Gly Pro Arg Val Val 1 5 10 15 Glu Arg His 68 18 PRT Homo sapiens 68 Asp Phe Leu Ala Glu Gly Gly Gly Val Arg Gly Pro Arg Val Val Glu 1 5 10 15 Arg His 69 17 PRT Homo sapiens 69 Phe Leu Ala Glu Gly Gly Gly Val Arg Gly Pro Arg Val Val Glu Arg 1 5 10 15 His 70 16 PRT Homo sapiens 70 Leu Ala Glu Gly Gly Gly Val Arg Gly Pro Arg Val Val Glu Arg His 1 5 10 15 71 15 PRT Homo sapiens 71 Ala Glu Gly Gly Gly Val Arg Gly Pro Arg Val Val Glu Arg His 1 5 10 15 72 14 PRT Homo sapiens 72 Glu Gly Gly Gly Val Arg Gly Pro Arg Val Val Glu Arg His 1 5 10 73 13 PRT Homo sapiens 73 Gly Gly Gly Val Arg Gly Pro Arg Val Val Glu Arg His 1 5 10 74 12 PRT Homo sapiens 74 Gly Gly Val Arg Gly Pro Arg Val Val Glu Arg His 1 5 10 75 1048 PRT Homo sapiens 75 Met Ala Phe Pro Pro Arg Arg Arg Leu Arg Leu Gly Pro Arg Gly Leu 1 5 10 15 Pro Leu Leu Leu Ser Gly Leu Leu Leu Pro Leu Cys Arg Ala Phe Asn 20 25 30 Leu Asp Val Asp Ser Pro Ala Glu Tyr Ser Gly Pro Glu Gly Ser Tyr 35 40 45 Phe Gly Phe Ala Val Asp Phe Phe Val Pro Ser Ala Ser Ser Arg Met 50 55 60 Phe Leu Leu Val Gly Ala Pro Lys Ala Asn Thr Thr Gln Pro Gly Ile 65 70 75 80 Val Glu Gly Gly Gln Val Leu Lys Cys Asp Trp Ser Ser Thr Arg Arg 85 90 95 Cys Gln Pro Ile Glu Phe Asp Ala Thr Gly Asn Arg Asp Tyr Ala Lys 100 105 110 Asp Asp Pro Leu Glu Phe Lys Ser His Gln Trp Phe Gly Ala Ser Val 115 120 125 Arg Ser Lys Gln Asp Lys Ile Leu Ala Cys Ala Pro Leu Tyr His Trp 130 135 140 Arg Thr Glu Met Lys Gln Glu Arg Glu Pro Val Gly Thr Cys Phe Leu 145 150 155 160 Gln Asp Gly Thr Lys Thr Val Glu Tyr Ala Pro Cys Arg Ser Gln Asp 165 170 175 Ile Asp Ala Asp Gly Gln Gly Phe Cys Gln Gly Gly Phe Ser Ile Asp 180 185 190 Phe Thr Lys Ala Asp Arg Val Leu Leu Gly Gly Pro Gly Ser Phe Tyr 195 200 205 Trp Gln Gly Gln Leu Ile Ser Asp Gln Val Ala Glu Ile Val Ser Lys 210 215 220 Tyr Asp Pro Asn Val Tyr Ser Ile Lys Tyr Asn Asn Gln Leu Ala Thr 225 230 235 240 Arg Thr Ala Gln Ala Ile Phe Asp Asp Ser Tyr Leu Gly Tyr Ser Val 245 250 255 Ala Val Gly Asp Phe Asn Gly Asp Gly Ile Asp Asp Phe Val Ser Gly 260 265 270 Val Pro Arg Ala Ala Arg Thr Leu Gly Met Val Tyr Ile Tyr Asp Gly 275 280 285 Lys Asn Met Ser Ser Leu Tyr Asn Phe Thr Gly Glu Gln Met Ala Ala 290 295 300 Tyr Phe Gly Phe Ser Val Ala Ala Thr Asp Ile Asn Gly Asp Asp Tyr 305 310 315 320 Ala Asp Val Phe Ile Gly Ala Pro Leu Phe Met Asp Arg Gly Ser Asp 325 330 335 Gly Lys Leu Gln Glu Val Gly Gln Val Ser Val Ser Leu Gln Arg Ala 340 345 350 Ser Gly Asp Phe Gln Thr Thr Lys Leu Asn Gly Phe Glu Val Phe Ala 355 360 365 Arg Phe Gly Ser Ala Ile Ala Pro Leu Gly Asp Leu Asp Gln Asp Gly 370 375 380 Phe Asn Asp Ile Ala Ile Ala Ala Pro Tyr Gly Gly Glu Asp Lys Lys 385 390 395 400 Gly Ile Val Tyr Ile Phe Asn Gly Arg Ser Thr Gly Leu Asn Ala Val 405 410 415 Pro Ser Gln Ile Leu Glu Gly Gln Trp Ala Ala Arg Ser Met Pro Pro 420 425 430 Ser Phe Gly Tyr Ser Met Lys Gly Ala Thr Asp Ile Asp Lys Asn Gly 435 440 445 Tyr Pro Asp Leu Ile Val Gly Ala Phe Gly Val Asp Arg Ala Ile Leu 450 455 460 Tyr Arg Ala Arg Pro Val Ile Thr Val Asn Ala Gly Leu Glu Val Tyr 465 470 475 480 Pro Ser Ile Leu Asn Gln Asp Asn Lys Thr Cys Ser Leu Pro Gly Thr 485 490 495 Ala Leu Lys Val Ser Cys Phe Asn Val Arg Phe Cys Leu Lys Ala Asp 500 505 510 Gly Lys Gly Val Leu Pro Arg Lys Leu Asn Phe Gln Val Glu Leu Leu 515 520 525 Leu Asp Lys Leu Lys Gln Lys Gly Ala Ile Arg Arg Ala Leu Phe Leu 530 535 540 Tyr Ser Arg Ser Pro Ser His Ser Lys Asn Met Thr Ile Ser Arg Gly 545 550 555 560 Gly Leu Met Gln Cys Glu Glu Leu Ile Ala Tyr Leu Arg Asp Glu Ser 565 570 575 Glu Phe Arg Asp Lys Leu Thr Pro Ile Thr Ile Phe Met Glu Tyr Arg 580 585 590 Leu Asp Tyr Arg Thr Ala Ala Asp Thr Thr Gly Leu Gln Pro Ile Leu 595 600 605 Asn Gln Phe Thr Pro Ala Asn Ile Ser Arg Gln Ala His Ile Leu Leu 610 615 620 Asp Cys Gly Glu Asp Asn Val Cys Lys Pro Lys Leu Glu Val Ser Val 625 630 635 640 Asp Ser Asp Gln Lys Lys Ile Tyr Ile Gly Asp Asp Asn Pro Leu Thr 645 650 655 Leu Ile Val Lys Ala Gln Asn Gln Gly Glu Gly Ala Tyr Glu Ala Glu 660 665 670 Leu Ile Val Ser Ile Pro Leu Gln Ala Asp Phe Ile Gly Val Val Arg 675 680 685 Asn Asn Glu Ala Leu Ala Arg Leu Ser Cys Ala Phe Lys Thr Glu Asn 690 695 700 Gln Thr Arg Gln Val Val Cys Asp Leu Gly Asn Pro Met Lys Ala Gly 705 710 715 720 Thr Gln Leu Leu Ala Gly Leu Arg Phe Ser Val His Gln Gln Ser Glu 725 730 735 Met Asp Thr Ser Val Lys Phe Asp Leu Gln Ile Gln Ser Ser Asn Leu 740 745 750 Phe Asp Lys Val Ser Pro Val Val Ser His Lys Val Asp Leu Ala Val 755 760 765 Leu Ala Ala Val Glu Ile Arg Gly Val Ser Ser Pro Asp His Ile Phe 770 775 780 Leu Pro Ile Pro Asn Trp Glu His Lys Glu Asn Pro Glu Thr Glu Glu 785 790 795 800 Asp Val Gly Pro Val Val Gln His Ile Tyr Glu Leu Arg Asn Asn Gly 805 810 815 Pro Ser Ser Phe Ser Lys Ala Met Leu His Leu Gln Trp Pro Tyr Lys 820 825 830 Tyr Asn Asn Asn Thr Leu Leu Tyr Ile Leu His Tyr Asp Ile Asp Gly 835 840 845 Pro Met Asn Cys Thr Ser Asp Met Glu Ile Asn Pro Leu Arg Ile Lys 850 855 860 Ile Ser Ser Leu Gln Thr Thr Glu Lys Asn Asp Thr Val Ala Gly Gln 865 870 875 880 Gly Glu Arg Asp His Leu Ile Thr Lys Arg Asp Leu Ala Leu Ser Glu 885 890 895 Gly Asp Ile His Thr Leu Gly Cys Gly Val Ala Gln Cys Leu Lys Ile 900 905 910 Val Cys Gln Val Gly Arg Leu Asp Arg Gly Lys Ser Ala Ile Leu Tyr 915 920 925 Val Lys Ser Leu Leu Trp Thr Glu Thr Phe Met Asn Lys Glu Asn Gln 930 935 940 Asn His Ser Tyr Ser Leu Lys Ser Ser Ala Ser Phe Asn Val Ile Glu 945 950 955 960 Phe Pro Tyr Lys Asn Leu Pro Ile Glu Asp Ile Thr Asn Ser Thr Leu 965 970 975 Val Thr Thr Asn Val Thr Trp Gly Ile Gln Pro Ala Pro Met Pro Val 980 985 990 Pro Val Trp Val Ile Ile Leu Ala Val Leu Ala Gly Leu Leu Leu Leu 995 1000 1005 Ala Val Leu Val Phe Val Met Tyr Arg Met Gly Phe Phe Lys Arg 1010 1015 1020 Val Arg Pro Pro Gln Glu Glu Gln Glu Arg Glu Gln Leu Gln Pro 1025 1030 1035 His Glu Asn Gly Glu Gly Asn Ser Glu Thr 1040 1045 76 788 PRT Homo sapiens 76 Met Arg Ala Arg Pro Arg Pro Arg Pro Leu Trp Val Thr Val Leu Ala 1 5 10 15 Leu Gly Ala Leu Ala Gly Val Gly Val Gly Gly Pro Asn Ile Cys Thr 20 25 30 Thr Arg Gly Val Ser Ser Cys Gln Gln Cys Leu Ala Val Ser Pro Met 35 40 45 Cys Ala Trp Cys Ser Asp Glu Ala Leu Pro Leu Gly Ser Pro Arg Cys 50 55 60 Asp Leu Lys Glu Asn Leu Leu Lys Asp Asn Cys Ala Pro Glu Ser Ile 65 70 75 80 Glu Phe Pro Val Ser Glu Ala Arg Val Leu Glu Asp Arg Pro Leu Ser 85 90 95 Asp Lys Gly Ser Gly Asp Ser Ser Gln Val Thr Gln Val Ser Pro Gln 100 105 110 Arg Ile Ala Leu Arg Leu Arg Pro Asp Asp Ser Lys Asn Phe Ser Ile 115 120 125 Gln Val Arg Gln Val Glu Asp Tyr Pro Val Asp Ile Tyr Tyr Leu Met 130 135 140 Asp Leu Ser Tyr Ser Met Lys Asp Asp Leu Trp Ser Ile Gln Asn Leu 145 150 155 160 Gly Thr Lys Leu Ala Thr Gln Met Arg Lys Leu Thr Ser Asn Leu Arg 165 170 175 Ile Gly Phe Gly Ala Phe Val Asp Lys Pro Val Ser Pro Tyr Met Tyr 180 185 190 Ile Ser Pro Pro Glu Ala Leu Glu Asn Pro Cys Tyr Asp Met Lys Thr 195 200 205 Thr Cys Leu Pro Met Phe Gly Tyr Lys His Val Leu Thr Leu Thr Asp 210 215 220 Gln Val Thr Arg Phe Asn Glu Glu Val Lys Lys Gln Ser Val Ser Arg 225 230 235 240 Asn Arg Asp Ala Pro Glu Gly Gly Phe Asp Ala Ile Met Gln Ala Thr 245 250 255 Val Cys Asp Glu Lys Ile Gly Trp Arg Asn Asp Ala Ser His Leu Leu 260 265 270 Val Phe Thr Thr Asp Ala Lys Thr His Ile Ala Leu Asp Gly Arg Leu 275 280 285 Ala Gly Ile Val Gln Pro Asn Asp Gly Gln Cys His Val Gly Ser Asp 290 295 300 Asn His Tyr Ser Ala Ser Thr Thr Met Asp Tyr Pro Ser Leu Gly Leu 305 310 315 320 Met Thr Glu Lys Leu Ser Gln Lys Asn Ile Asn Leu Ile Phe Ala Val 325 330 335 Thr Glu Asn Val Val Asn Leu Tyr Gln Asn Tyr Ser Glu Leu Ile Pro 340 345 350 Gly Thr Thr Val Gly Val Leu Ser Met Asp Ser Ser Asn Val Leu Gln 355 360 365 Leu Ile Val Asp Ala Tyr Gly Lys Ile Arg Ser Lys Val Glu Leu Glu 370 375 380 Val Arg Asp Leu Pro Glu Glu Leu Ser Leu Ser Phe Asn Ala Thr Cys 385 390 395 400 Leu Asn Asn Glu Val Ile Pro Gly Leu Lys Ser Cys Met Gly Leu Lys 405 410 415 Ile Gly Asp Thr Val Ser Phe Ser Ile Glu Ala Lys Val Arg Gly Cys 420 425 430 Pro Gln Glu Lys Glu Lys Ser Phe Thr Ile Lys Pro Val Gly Phe Lys 435 440 445 Asp Ser Leu Ile Val Gln Val Thr Phe Asp Cys Asp Cys Ala Cys Gln 450 455 460 Ala Gln Ala Glu Pro Asn Ser His Arg Cys Asn Asn Gly Asn Gly Thr 465 470 475 480 Phe Glu Cys Gly Val Cys Arg Cys Gly Pro Gly Trp Leu Gly Ser Gln 485 490 495 Cys Glu Cys Ser Glu Glu Asp Tyr Arg Pro Ser Gln Gln Asp Glu Cys 500 505 510 Ser Pro Arg Glu Gly Gln Pro Val Cys Ser Gln Arg Gly Glu Cys Leu 515 520 525 Cys Gly Gln Cys Val Cys His Ser Ser Asp Phe Gly Lys Ile Thr Gly 530 535 540 Lys Tyr Cys Glu Cys Asp Asp Phe Ser Cys Val Arg Tyr Lys Gly Glu 545 550 555 560 Met Cys Ser Gly His Gly Gln Cys Ser Cys Gly Asp Cys Leu Cys Asp 565 570 575 Ser Asp Trp Thr Gly Tyr Tyr Cys Asn Cys Thr Thr Arg Thr Asp Thr 580 585 590 Cys Met Ser Ser Asn Gly Leu Leu Cys Ser Gly Arg Gly Lys Cys Glu 595 600 605 Cys Gly Ser Cys Val Cys Ile Gln Pro Gly Ser Tyr Gly Asp Thr Cys 610 615 620 Glu Lys Cys Pro Thr Cys Pro Asp Ala Cys Thr Phe Lys Lys Glu Cys 625 630 635 640 Val Glu Cys Lys Lys Phe Asp Arg Glu Pro Tyr Met Thr Glu Asn Thr 645 650 655 Cys Asn Arg Tyr Cys Arg Asp Glu Ile Glu Ser Val Lys Glu Leu Lys 660 665 670 Asp Thr Gly Lys Asp Ala Val Asn Cys Thr Tyr Lys Asn Glu Asp Asp 675 680 685 Cys Val Val Arg Phe Gln Tyr Tyr Glu Asp Ser Ser Gly Lys Ser Ile 690 695 700 Leu Tyr Val Val Glu Glu Pro Glu Cys Pro Lys Gly Pro Asp Ile Leu 705 710 715 720 Val Val Leu Leu Ser Val Met Gly Ala Ile Leu Leu Ile Gly Leu Ala 725 730 735 Ala Leu Leu Ile Trp Lys Leu Leu Ile Thr Ile His Asp Arg Lys Glu 740 745 750 Phe Ala Lys Phe Glu Glu Glu Arg Ala Arg Ala Lys Trp Asp Thr Ala 755 760 765 Asn Asn Pro Leu Tyr Lys Glu Ala Thr Ser Thr Phe Thr Asn Ile Thr 770 775 780 Tyr Arg Gly Thr 785 

1. A screening for the identification of agents which modulate the interaction of the fibrinogen E fragment, or peptide derivative thereof, with a vitronectin receptor wherein said method comprises the steps of: i) providing a polypeptide comprising the amino acid sequence presented in FIG. 2, or active binding fragment thereof; ii) providing at least one peptide comprising an amino acid sequence selected from the sequences presented in FIG. 1; iii) providing at least one agent to be tested; iv) forming a preparation of (i), (ii) and (iii); and v) detecting or measuring the effect of the agent in (iii) on the interaction of the peptide and polypeptide in (i) and (ii).
 2. A method according to claim 1 wherein said agent is pre-incubated with polypeptide in (i) prior to addition of the peptide in (ii).
 3. A method according to claim 1 wherein said agent is pre-incubated with the peptide in (ii) prior to addition of the agent.
 4. A method according to claim 1 wherein the peptide in (ii) comprises an amino acid sequence consisting of the sequence: XXXXXLXEXXGXXXPRVXXR.


5. A method according to claim 1 wherein the peptide in (ii) comprises an amino acid sequence consisting of the sequence: SXXXXXLXEXXGXXXPRVXXR.


6. A method according to claim 1 wherein the peptide in (ii) comprises an amino acid sequence consisting of the sequence: XXXXXLXEXXGXXXPRVVXR.


7. A method according to claim 1 wherein the peptide in (ii) comprises an amino acid sequence consisting of the sequence: GEGXFLXEXXGXXXPRVVXR.
 8. A method according to claim 7 wherein the peptide in (ii) comprises an amino acid sequence selected from the group consisting of: GEGDFLAEGGGVRGPRVVER GEGDFLAEGGGXRGPRVVER GEGDFLAEGGGVXGPRVVER GEGDFLAEGGGVRXPRVVER GEGDFLAEGGGVRGPRVXER GEGDFLAEGGGVRGPRVVXR GEGDFLAEGGGXXXPRVVXR GEGDFLAEGGGXXXPRVXXR

wherein X is any amino acid residue.
 9. A method according to claim 1 wherein the peptide in (ii) is selected from the group consisting of: AXSXXXDFLAXGGGVXXPXV VXXH ADSGEGDFLAEGGGVRGPRV VEXH ADSGEGDFLAXGGGVRGPRV VERH ADSGEGDFLA EGGGVXGPRV VERH ADSGEGXFLA EGGGVRGPRV VERH AXSGXGDFLA EGGGVRGPRV VERH ADSGXGDFLA EGGGVRGPRV VERH AXSGEGDFLA EGGGVRGPRV VERH ADSGEGDFLA EGGGVRGPRV VXRH

wherein X is any amino acid residue.
 10. A method according to claim 1 wherein X is selected from the group consisting of alanine, valine, leucine, isoleucine, or proline.
 11. A method according to claim 10 wherein X is alanine.
 12. A method according to claim 1 wherein the peptide in (ii) comprises an amino acid sequence consisting of: XFLAEGGGVXG wherein X is any amino acid residue.
 13. The method of claim 12 wherein wherein the N-terminal X is an acidic amino acid and the C-terminal X is a basic amino acid or wherein the N-terminal X is a basic amino acid and the C-terminal X is an acidic amino acid.
 14. The method of claim 13 wherein wherein the N-terminal X is D and the C-terminal X is R.
 15. A method according to claim 1 wherein the vitronectin receptor is soluble.
 16. A method according to claim 1 wherein the vitronectin receptor is presented by a cell.
 17. A method according to claim 16 wherein said cell naturally expresses the vitronectin receptor.
 18. A method according to claim 16 wherein said cell is selected from the group consisting of: endothelial cells, smooth muscle cells, osteoclasts and tumour cells.
 19. A method according to claim 18 wherein said cell is an endothelial cell.
 20. A method according to claim 16 wherein said cell does not naturally express the vitronectin receptor.
 21. A method according to claim 20 wherein said cells are genetically engineered to express the vitronectin receptor.
 22. An agent(s) obtainable by the screening method according to claim
 1. 23. An agent according to claim 22 wherein the agent is a foldamer, low molecular weight compound, peptide, peptide derivative or polypeptide.
 24. An agent according to claim 23 wherein said agent is an antibody.
 25. An agent according to claim 24 wherein said agent is a monoclonal antibody.
 26. An agent according to claim 25 wherein said agent is a chimaeric antibody.
 27. An agent according to claim 25 wherein said agent is a humanized antibody.
 28. A screening method for the identification of agents with vitronectin binding activity which have the capability to modulate angiogenesis comprising the steps of: i) providing a member of the integrin family which binds the peptide α1-24, or variant thereof; ii) providing at least one candidate binding agent; iii) forming a preparation comprising a combination of (i) and (ii); iv) detecting or measuring the binding of the agent in (ii) with the polypeptide in (i); and optionally iv) testing the capability of the agent to modulate angiogenesis.
 29. A method according to claim 28 wherein the agent with vitronectin receptor binding activity has anti-angiogenic activity.
 30. A method according to claim 28 wherein an agent with vitronectin receptor binding activity has pro-angiogenic activity.
 31. A method according to claim 28 wherein the vitronectin receptor is αvβ3 integrin and comprises the amino acid sequence as represented in FIG.
 2. 32. A method according to claim 28 wherein said vitronectin receptor is soluble.
 33. A method according to claim 28 wherein said vitronectin receptor is presented by a cell.
 34. A method according to claim 33 wherein said cell naturally expresses a vitronectin receptor.
 35. A method according to claim 33 wherein said cell is selected from the group consisting of the following cell-types: endothelial cells, smooth muscle cells, osteoclasts and tumour cells.
 36. A method according to claim 35 wherein said cell is an endothelial cell.
 37. A method according to claim 33 wherein said cell does not naturally express vitronectin receptor.
 38. A method according to claim 37 wherein said cell is genetically engineered to express the vitronectin receptor.
 39. An agent obtainable by the method according to claim
 28. 40. An agent according to claim 39 wherein said agent has anti-angiogenic activity.
 41. An agent according to claim 39 wherein said agent has pro-angiogenic activity.
 42. An agent according to claim 39 wherein the agent is a polypeptide. 